Process for automated production of synchronous mammalian cells

ABSTRACT

A process for automatically producing relatively large, biochemically balanced populations of synchronous mammalian cells without requiring the presence of an investigator for prolonged periods of time, which includes subculturing the cells in a medium inside roller bottles, allowing growth of the cells, and then rotating the bottles at a speed sufficient to detach rounded mitotic cells and collecting the cells so detached.

Unite States Patent [1 1 Klevecz Mar. 18, 1975 l l PROCESS FOR AUTOMATEDPRODUCTION OF SYNCHRONOUS MAMMALIAN CELLS [76] Inventor: Robert R.Klevecz, 227 Bettyhill Dr., Duarte, Calif. 91010 221 Filed: May 14,1973

[21 Appl. No.: 360,047

[52] US. Cl. 195/l.8 [51] Int. Cl Cl2k 9/00 [58] Field of Search195/127, 1.8

[56] References Cited UNITED STATES PATENTS 3 7l 1,379 l/l973 Adamsl95/l27 OTHER PU BLICATIONS WillmerCells & Tissues in Culture, Vol.(1966), p. 91.

Primary Examiner-Sam Rosen Attorney, Agent, or Firm-John Joseph HallABSTRACT 9 Claims, 1 Drawing Figure PROCESS FOR AUTOMATED PRODUCTION OFSYNCHRONOUS MAMMALIAN CELLS BACKGROUND OF THE INVENTION 1. Field of theInvention This invention relates to the production of synchronousmammalian cells.

2. Prior Art Applicant is unaware of any prior art disclosing a processfor automated production of synchronous mammalian cells without chemicaltreatment or lowered temperatures and which dispenses with the presenceof the investigator for prolonged periods of time. Chemical inhibitorswhich align or synchronize cells only do so with respect to a singlebiochemical parameter and thus may detrimentally affect the resultingcell population. Alignment or synchronization methods with singleinhibitors or a combination of inhibitors may alter normal cellmetabolism and may introduce artifactual perturbations or obscurenatural periodicities. Further, simple mitotic selection techniquesusually result in low yields, besides requiring the investigatorspresence for long periods of time.

SUMMARY OF THE INVENTION The process for automated production ofsynchronous mammalian cells uses roller bottles containing cell mediumwhich has been incubated for 30 minutes. Mammalian cells are thensubcultured for about 24 hours in the roller bottles. Then, the rollerbottles are speeded up sufficiently for a given period oftime todislodge rounded mitotic cells in the medium. The detached cells inmedium are pumped from the roller bottles and collected in a manifold,and then discharged into a growth flask.

The process is then repeated at the desired time interval to suit thecell cycle time and the particular requirements of the investigator. Allof these steps of the process are automatically carried out by suitableapparatus of a conventional type, without the prolonged presence of theinvestigator.

It is, therefore, an object of this invention to provide a process forautomated production of synchronous mammalian cells which has no needfor the presence of the investigator for prolonged periods of time.

Another object of this invention is to provide a process for automatedproduction of synchronous mammalian cells without the use of chemicaltreatment, such as inhibitors.

A further object ofthis invention is to provide a process for automatedproduction of synchronous mammalian cells in relatively high yields.

A yet further object of this invention is to provide a process forautomated production of biochemically balanced populations ofsynchronous mammalian cells, without the use of altered medium orlowered temperatures.

These and other objects will be more readily understood by reference tothe following description and claims, taken in conjunction with theaccompanying drawing illustrating a preferred sequence of operations ofthe process.

The following Example 1 is an embodiment of the invention using Chinesehamster cells, known as cell line V 79. The medium for the cells isMcCoys 5a medium supplemented with fetal calf serum and 100 mg/lneomycin.

EXAMPLE I The entire cycle of the mammalian cell, Chinese hamster cellline V79, with a 12-hour generation time, is examined.

A series of roller bottles is set up in a standard Bellco model 7400roller apparatus modified to operate at increased revolutions by addinga one-way clutch'and a fractional horsepower bodine motor. The rollerbottles are incubated'each with about 25 ml. of the medium for about 30minutes at 37 C. Then, cells are subcultured,

i.e., inoculated in the medium, at a density of 3 X 10 7 Then, theroller apparatus is speeded up to a speed of about rpm for about 5minutes, R thereby causing the dislodging of rounded mitotic cells,which are selectively detached in the medium in the roller bottles.

The detached cells in the medium are collected through manifold M anddischarged into a growth flask 0,, through valve V However, the firstbatch of detached cells is always discarded because it is contaminatedwith cells which have never attached or were loosely attached but werenon-mitotic.

The growth flasks are selected by the opening and closing of a series ofvalves V,,, V, through V the number of valves and flasks may be variedas desired.

This first cycle may be called the purge cycle.

Next, a fresh 25 ml. charge of medium is then pumped into the rollerbottles by pump P,- while valve V remains open to allow any medium andcells trapped in the line to drain into a waste receptacle. The slowroller speed is repeated for about 55 minutes, and then the fast speedfor about. 5 minutes.

This time, the detached cells are pumped by pump P out of the rollerbottles and collected in the manifold M, and, by suitable valve timingthrough valve V discharged into growth flask (3,.

Throughout the process, the detached cells are kept at 37 C.

The process is then repeated every hour for 12 hours automatically, sothat detached cells will be pumped and collected through the manifoldand valves selectively into growth flasks G through G by valves Vthrough V Each of the growth flasks thus has a selected synchronousculture separated in the cell cycle from the others by 1 hour. Theprogram is automatic and has no need for the presence of theinvestigator during this 12-hour period.

The speed of the roller can be varied to suit the particular cell cycletime. Also, the slow speed of the roller may be set from 20 to minutesin l-minute increments.

Similarly, the fast turning of the roller may be varied as desired withrespect to time of fast turning, prior to slow turning.

EXAMPLE 2 The process of Example 1 was repeated, except for a change inthe slow and fast rotation of the rollers.

The program controller was adjusted to provide a slow turning speed ofabout 0.5 rpm of the roller bottles for a period of 10 minutes.Thereafter, the fast turning speed of about 100 rpm for the rollerbottles was set for 50 minutes, and the detached cells were dischargedinto the growth flasks.

The quality of synchrony using the processes of Examples l and 2 wascompared with that obtained by conventional manual selection of cells.The process of Example 1 produced about the same yield of cells persample as by manual selection, but the process of Example 2 produced ayield of over three times the yield obtained by manual selection.

Yields were tested by conventional methods of counting in a Coulterelectronic cell counter and by centrifuging and fixing for cytologicalanalysis.

I have found that the process produces successful results using humancells, as indicated in the following Example.

EXAMPLE 3 This Example used a normal diploid human cell line known asWl-38, human fibroblasts. Wl-38 cells were brought into a synchronousstate by repeated selection and automated detachment of mitotic cellsfrom roller bottles according to the process of Example 1, except thatthese cells were subcultured at a density of 5 X cells per roller. Also,the program was set for a 24-hour period and used 24 growth flasks.

The process of Example 3 produced a relatively large yield ofsynchronous Wl-38 cells, about 0.5 X 10 cells per growth flask.

It is known that standard methods such as colcemidenhanced mitoticselection and S phase arrest have failed. Also, simple mitotic selectionyielded only a vanishingly small population of cells.

Although I have described my invention in detail with reference to theaccompanying drawing illustrating a preferred embodiment of theinvention in conjunction with the specification and claims, it isunderstood that numerous changes in the details of construction andarrangement of parts and components may be made without departing fromthe spirit and scope of the invention as hereinafter claimed.

1 claim:

1. A process for automated production of synchronous mammalian cells,comprising:

adding a suitable medium to a seriesof roller bottles in a rollerapparatus;

incubating the medium;

subculturing the medium with mammalian cells;

allowing growth of the cells for about 24 hours;

initiating turning of the roller bottles by automatic controllers at aspeed sufficiently slow to prevent detaching said cells;

speeding up the turning of the roller bottles by automatic controllersto a speed sufficient to detach rounded mitotic cells;

removing the detached rounded mitotic cells from the roller bottles;

Collecting the detached rounded mitotic cells in a manifold; and

, discharging said cells into a growth flask through a valve.

2. A process according to claim 1 which the period of time forinitiating turning of the roller bottles is about 55 minutes.

3. A process according to claim 1 in which the period of time forspeeding up the turning of the bottles is about 5 minutes.

4. A process according to claim 1 in which the initiating turning of therollers is about 0.5 rpm.

5. A process according to claim 1 in which the speeded up turning of therollers is about rpm.

6. A process for automated production of synchronous mammalian cells,comprising:

adding a suitable medium to a series of roller bottles in a rollerapparatus;

incubating the medium for about 30 minutes;

subculturing the medium with mammalian cells;

allowing growth of the cells for about 24 hours;

initiating turning of the roller bottles by automatic controllers forabout 55 minutes at about 0.5 rpm;

speeding up the turning of the roller bottles by automatic controllersfor about 5 minutes at about 100 rpm to detach rounded mitotic cells;

removing the detached rounded mitotic cells from the roller bottles;

collecting the detached rounded mitotic cells in a manifold; and

discharging said cells into a growth flask through a valve.

7. A process for automated production of synchronous mammalian cells,comprising:

adding a suitable medium to a series of roller bottles in roller.apparatus;

incubating the medium for about 30 minutes;

subculturing the medium with mammalian cells;

allowing grown of the cells for about 24 hours.

initiating turning of the roller bottles by automatic controllers forabout 10 minutes at about 0.5 rpm;

speeding up the turning of the roller bottles by automatic controllersfor about 50 minutes at about 100 rpm to detach rounded mitotic cells;

removing the detached rounded mitotic cells from the roller bottles;

collecting the detached rounded mitotic cells in a manifold; and

discharging said cells into a growth flask through a valve.

8. A process for automated production of synchronous mammalian cells,comprising:

subculturing mammalian cells in a suitable medium in a containerrotatable around its axis;

allowing growth of said cells for about 24 hours;

causing relatively rapid rotation of said container by automaticcontrollers for a pre-determined period of time at a speed sufficient todetach rounded mitotic cells; and

collecting the detached rounded mitotic cells in a container.

9. A process for automated production of synchronous mammalian cells,comprising:

automatically pumping a charge of medium into a container rotatablearound its axis;

subculturing mammalian cells in said medium for about 24 hours while thecontainer is rotating at a relatively slow speed;

causing relatively rapid rotation of said container by collecting thedetached rounded mitotic cells in a automatic controllers for apre-determined period container by the automatic opening and closing ofof time at a speed sufficient to detach rounded mia valve means. toticcells; and

1. A PROCESS FOR AUTOMATED PRODUCTION OF SYNCHRONOUS MAMMALIAN CELLS,COMPRISING: ADDING A SUITABLE MEDIUM TO A SERIES OF ROLLER BOTTLES IN AROLLER APPARATUS; INCUBATING THE MEDIUM; SUBCULTURING THE MEDIUM WITHMAMALIAN CELLS; ALLOWING GROWTH OF THE CELLS FOR ABOUT 24 HOURS;INITIATING TURNING OF THE ROLLER BOTTLES BY AUTOMATIC CONTROLLERS AT ASPEED SUFFICIENTLY SLOW TO PREVENT DETACHING SAID CELLS; SPEEDING UP THETURNING OF THE ROLLER BOTTLES BY AUTOMATIC CONTROLLERS TO A SPEEDSUFFICIENT TO DETACH ROUNDED MITOTIC CELLS; REMOVING THE DETACHEDROUNDED MITOTIC CELLS FROM THE ROLLER BOTTLES; COLLECTING THE DETACHEDROUNDED MITOTIC CELLS IN A MANIFOLD; AND DISCHARGING SAID CELLS INTOGROWTH FLASK THROUGH A VALVE.
 2. A process according to claim 1 whichthe period of time for initiating turning of the roller bottles is about55 minutes.
 3. A process according to claim 1 in which the period oftime for speeding up the turning of the bottles is about 5 minutes.
 4. Aprocess according to claim 1 in which the initiating turning of therollers is about 0.5 rpm.
 5. A process according to claim 1 in which thespeeded up turning of the rollers is about 100 rpm.
 6. A process forautomated production of synchronous mammalian cells, comprising: addinga suitable medium to a series of roller bottles in a roller apparatus;incubating the medium for about 30 minutes; subculturing the medium withmammalian cells; allowing growth of the cells for about 24 hours;initiating turning of the roller bottles by automatic controllers forabout 55 minutes at about 0.5 rpm; speeding up the turning of the rollerbottles by automatic controllers for about 5 minutes at about 100 rpm todetach rounded mitotic cells; removing the detached rounded mitoticcells from the roller bottles; collecting the detached rounded mitoticcells in a manifold; and discharging said cells into a growth flaskthrough a valve.
 7. A process for automated production of synchronousmammalian cells, comprising: adding a suitable medium to a series ofroller bottles in roller apparatus; incubating the medium for about 30minutes; subculturing the medium with mammalian cells; allowing grown ofthe cells for about 24 hours. initiatIng turning of the roller bottlesby automatic controllers for about 10 minutes at about 0.5 rpm; speedingup the turning of the roller bottles by automatic controllers for about50 minutes at about 100 rpm to detach rounded mitotic cells; removingthe detached rounded mitotic cells from the roller bottles; collectingthe detached rounded mitotic cells in a manifold; and discharging saidcells into a growth flask through a valve.
 8. A process for automatedproduction of synchronous mammalian cells, comprising: subculturingmammalian cells in a suitable medium in a container rotatable around itsaxis; allowing growth of said cells for about 24 hours; causingrelatively rapid rotation of said container by automatic controllers fora pre-determined period of time at a speed sufficient to detach roundedmitotic cells; and collecting the detached rounded mitotic cells in acontainer.
 9. A process for automated production of synchronousmammalian cells, comprising: automatically pumping a charge of mediuminto a container rotatable around its axis; subculturing mammalian cellsin said medium for about 24 hours while the container is rotating at arelatively slow speed; causing relatively rapid rotation of saidcontainer by automatic controllers for a pre-determined period of timeat a speed sufficient to detach rounded mitotic cells; and collectingthe detached rounded mitotic cells in a container by the automaticopening and closing of a valve means.